A rapid PCR-based method for the identification of ob mutant mice.
With increasing incidence of obesity, there is greater demand for suitable research and therapeutic models. The ob/ob mouse model develops obesity by 5 weeks of age. Previously, a method using DNA purification, PCR, and restriction digestion of products was devised to identify mice bearing the ob allele. Here, we describe a direct PCR method that requires no DNA purification. Wild-type and ob-specific primers are used under the same conditions in two separate and simultaneously run three-primer PCRs. Standard PCR using the wild-type primer mix produces 191 bp and 104 bp bands in +/+ and ob/+ and only the control 191 bp band in ob/ob animals. The ob-specific primer reaction produces 191 bp and 123 bp bands in ob/+ and ob/ob and only the control 191 bp band in +/+ animals. Phenotypic weight gain in offspring of heterozygous intercrosses was used to validate genotypes. This primer-specific PCR method allows simultaneous identification of +/+, ob/+, and ob/ob genotypes prior to breeding age to facilitate breeding and research studies in an important model of clinical obesity.
Ellett
Justin D
JD
Department of Surgery, Division of Transplant, Medical University of South Carolina, Charleston, South Carolina, USA.
Evans
Zachary P
ZP
Zhang
Guojing
G
Chavin
Kenneth D
KD
Spyropoulos
Demetri D
DD
D000483:Alleles; D000818:Animals; D004247:DNA / Q000235:genetics; D004195:Disease Models, Animal; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005838:Genotype; D051379:Mice; D008820:Mice, Obese / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009765:Obesity / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA